The project goal is to understand the transition metal binding specificity of metalloregulatory proteins. The practical applications of this endeavor are at least two-fold: 1) several metalloregulatory proteins currently under investigation come from antibiotic-resistant pathogenic bacteria. Since no human analogue of these regulators exist, targeting them for drug therapy offers an attractive alternative to current antibiotic regimes. 2) Approximately 70% of the proteins in the human body bind metals. A firm understanding of the metal-binding site and hence the selection mechanism will be a huge first step in understanding the function of all metallo-proteins. To this end, we have chosen the Mn2+-dependent regulator MntR from Bacillus subtilis as our model of study. With metal-free starting material in hand, biochemical and biophysical characterization will proceed. The biochemical will focus on the native structural state of the protein (monomer/dimmer), metal-residue contacts, and protein/DNA interaction to name a few. The biophysical will include probing the metal-binding site with Electron Paramagnetic Resonance (EPR) spectroscopy, measuring the binding affinity of the regulator to DNA using Isothermal Titration Calorimetry (ITC) and studying the solution structure of the protein using Analytical Uitracentrifugation. With these tools and more at our disposal, we are confident that a more thorough understanding of the transition metal switch will be found.